The structural response of the cornea to changes in stromal hydration

Abstract

The primary aim of this study was to quantify the relationship between corneal structure and hydration in humans and pigs. X-ray scattering data were collected from human and porcine corneas equilibrated with polyethylene glycol (PEG) to varying levels of hydration, to obtain measurements of collagen fibril diameter, interfibrillar spacing (IFS) and intermolecular spacing. Both species showed a strong positive linear correlation between hydration and IFS² and a nonlinear, bi-phasic relationship between hydration and fibril diameter, whereby fibril diameter increased up to approximately physiological hydration, H = 3.0, with little change thereafter. Above H = 3.0, porcine corneas exhibited a larger fibril diameter than human corneas (p < 0.001). Intermolecular spacing also varied with hydration in a bi-phasic manner but reached a maximum value at a lower hydration (H = 1.5) than fibril diameter. Human corneas displayed a higher intermolecular spacing than porcine corneas at all hydrations (p < 0.0001). Human and porcine corneas required a similar PEG concentration to reach physiological hydration, suggesting that the total fixed charge that gives rise to the swelling pressure is the same. The difference in their structural responses to hydration can be explained by variations in molecular cross-linking and intra/interfibrillar water partitioning.

Keywords: collagen; cornea; fixed charge; hydration; proteoglycans; swelling.

Figure 1.

Schematic of corneal multi-modal X-ray scattering. X-rays are directed perpendicular to the corneal plane. The resulting X-ray scatter pattern is collected on a detector positioned behind the specimen. The distribution of small-angle X-ray scatter provides information about collagen parameters at the fibrillar level, whereas the wide-angle X-ray scatter provides information at the molecular level. (Online version in colour.)

Figure 2.

Analysis of X-ray scattering data. (a) The small-angle X-ray scatter pattern, which is circumferentially integrated to give a radial intensity profile. (b) Background-subtracted intensity profile (blue line), showing interference function peak (green circle) which provides a measure of the centre-to-centre separation distance of collagen fibrils. The fitted cylinder transform peak (Bessel function shown as a red line in b and inset c), which sits below the sharp third-order meridional reflection from corneal collagen, quantifies the average fibril diameter. (d) The wide-angle X-ray scatter pattern, which is circumferentially integrated around the collagen intermolecular reflection to give a radial intensity profile. (e) Radial intensity profile (red line), with fitted background function (green line). (f) The background-subtracted position of the intermolecular peak, once calibrated, gives a measure of the average collagen intermolecular separation.

Figure 3.

The relationship between the concentration of PEG in the bathing medium and the equilibrated stromal hydration in each species. Data for porcine, ovine and bovine corneas are shown as average values with standard deviation bars based on n = 5 at each data point. Data for human corneas represent a single specimen at each concentration. See the electronic supplementary material, S2 for tabulated data format.

Figure 4.

Structural transformation of the human and porcine corneal stroma with PEG adjusted hydration. The relationship between hydration and the square of the corneal collagen Bragg interfibrillar spacing (IFS²) (a), fibril diameter (b) and intermolecular spacing (IMS) (c) for PEG-equilibrated human and porcine corneas. See the electronic supplementary material, S3 for tabulated data format.

Figure 5.

Structural transformation of the porcine corneal stroma during air drying and equilibration. The relationship between hydration and the square of the corneal collagen Bragg interfibrillar spacing (IFS²) (a), fibril diameter (b) and intermolecular spacing (IMS) (c) for air-dried corneas and PEG-equilibrated corneas. See electronic supplementary material S4 for tabulated data format.

Figure 6.

Schematic of three collagen fibrils in cross-section (black) with associated glycosaminoglycans (GAGs; grey). For the purposes of this paper, the GAGs have been divided into two components: fibril coating GAGs and interstitial GAGs. X-ray scattering measurements of fibril diameter include the cylindrical collagen fibril plus the surrounding fibril coating (shown as a broken black line).

Figure 7.

Fibril diameter data (from figure 4) and intermolecular spacing² (IMS²) are shown at low hydrations, where the behaviour is linear. Intermolecular spacings are normalized to match the value of the corresponding fibril diameter at H = 0 for human (c) and pig (d) corneas, based upon the assumption that the thickness of the GAG coating is negligible when the fibrils are dry. A best-fit polynomial has been applied to each dataset.